Glutathione reductase (GR) is a homodimeric flavoenzyme that regenerates reduced glutathione, essential for cellular redox balance, with an active site cleft at the upper dimer interface. However, the role of intramolecular interactions within each monomer in maintaining the dimer interface and influencing enzyme activity remains unclear. This study investigates how hydrophobic interactions within each monomer near the dimer interface, particularly involving L416 (α12 helix) and F443 (α14 helix), affect the stability and activity of GR from the psychrotrophic bacterium